Derivation, characterization and therapeutic use of stem cells is among the most rapidly-developing fields of modern biology and medicine. Ability of stem cells differentiate into many lineages, as well as significantly influence processes of tissue regeneration paves the way for wide use of stem cell-based therapies in many medical applications. Multiple types of stem cells, embryonic and adult, are currently investigated. Though embryonic and induced pluripotent stem cells may be differentiated into any other cell types, their ability to form teratomas currently precludes their use in medical practice. Adult stem cells are currently used in humans, though only hematopoietic stem cells have been proven to provide therapeutic effect. One of the main problems is the difficulty in obtaining sufficient amount of stem cells, maintaining their “stemness” potential while in culture. Therefore, supply of stem cells is very limited, and novel sources are in high demand.
Human embryonic stem cells are derived from the pre-implantation or peri-implantation embryo and are characterized by the following major features: prolonged undifferentiated proliferation and stable developmental potential to form all the three of germ layers even after prolonged culture. Prolonged undifferentiated proliferation of human ECS is achieved by growing these cells on mouse embryonic fibroblast feeder layers in special ECS medium. Other specific features of human ESC, including: 1) high levels of telomerase activity; 2) formation of embryonic bodies; 3) expression of specific cellular markers Oct-4, Nanog, Sox-2 and surface markers CD117, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and alkaline phosphatase activity; and 4) formation of teratomas in immuno-compromised animals, are used to confirm derivation of pluripotent stem cell lines, similar to ECS.
Mesenchymal stem cells (MSC) are derived from adult body. Currently the most widely used and well characterized source of MSC is bone marrow. The basic features of adult mesenchymal stem cells (MSC) are: 1) proliferation potential; 2) the ability to differentiate; 3) plasticity; and 4) the ability to adhere to plastic, promoting the basic procedure for the MSC isolation, storage and usage. The MSC are characterized by expression of many common molecules required for adherence to solid surface and cell to cell interactions (Dominici et al., 2005). MSC possess a rich differential potential allowing them to differentiate into almost all possible mesenchymal lineages except hematopoietic cells. Several technical difficulties exist in practical approaches for obtaining stem cell populations from different organs of adult tissue due to very low occurrence of stem cells (fractions of a percent) and clear ethical problems associated with embryos or fetuses. Harvesting stem cells from adult sources such as bone marrow is labor-intensive and expensive. Harvesting stem cells from an adult donor, which is a current practice for obtaining hematopoietic cells and stromal cells of bone marrow possesses certain threats to donor, is expensive, labor-intensive and effective transplantation requires immunological match between donor and recipient.
Placenta, as a discarded material after birth, represents a unique opportunity for harvest of autologous or allogeneic populations of stem cells. Stem cells derived from feto-placental complex have been previously described. Stem cells of amniotic fluid have been described in many studies (Alviano et al, 2007; in't Anker et al, 2004). Stem cells isolated from human-term amniotic fluid may be cultured over 50 population doublings (in'tAnker et al, 2004). Phenotype of these cells was close to phenotype of MSC from bone marrow by expression of CD90, CD105, CD166, CD49, SH3, SH4 and negative for CD31, CD34, CD45, HLA-DR. However, at third trimester of pregnancy the viability of these cells decreases substantially and only in 20% it was possible to obtain viable stem cells from amniotic fluid. Embryonic-like stem cells in amniotic fluid are identified by expression of Oct-4, stem cell factor (SCF), high telomerase activity. Report by DeCoppi et al (2007) describes stem cell lines isolated from amniotic fluid following enrichment of c-kit positive cells. These cells expand without feeders up to 250 population doublings, are not tumorigenic, and can be differentiated into cell types representing cells of all the germ layers.
Yet another source of MSC is human placenta itself (Fukuchi Y et al, 2004; Matikainen T, Laine J. 2005). Previous studies (Linjuy Yen et al, 2005) demonstrated that multipotent, multilineage cells were present in the human term placenta. Although the initial cell culture consisted of both fibroblastoid and non-fibroblastoid cell types, only the fibroblastoid population remained after enzymatic digestion and passaging. MSC markers such as CD105/endoglin/SH-2 and SH-3, SH-4, integrins and matrix receptors displayed by placental stem cells, suggesting that these cells resemble MSC from the bone marrow. This is further supported by their fibroblastoid morphology, plastic-adherence nature, and mesodermal differentiation capabilities. In addition to MSC markers, cell surface markers SSEA-4, TRA-1-60, and TRA-1-81 were present on placenta-derived cells, which were previously found only in embryonic stem and germ cells.
First International Workshop on placenta-derived stem cells (Parolini et al, 2007) suggested the following nomenclature for cells derived from fetal placenta: Amniotic Epithelial Cells (hAEC), Amniotic mesenchymal Stromal cells (hAMSC), Chorionic Mesenchymal Stromal Cells (hCMSC), and Chorionic Trophoblastic Cells (hCTC) and suggested that cells isolated from fetal membranes should be termed mesenchymal stromal cells. Minimal criteria for defining these cells were adherence to plastic, formation of colonies, specific pattern of surface antigen expression and differentiation potential. hAEC were extensively studied by the group of S. Strom, (Miki et al, 2005, 2007). Cells obtained from amniotic membrane are phenotypically heterogeneous, could proliferate up to 5-10 passages and express very low levels of HLA-A,B,C. These cells express markers of pluripotent cells such as SSEA-3 and -4, TRA-1-60 and TRA-1-81, Oct-4, SOX-2 and Nanog. Such hAMC are capable to differentiate into the cells of all three germ layers (Tamagawa et al, 2004, Ilancheran et al, 2007). According to reports (Chien et al, 2006; Miki et al 2005; Sakuragawa et al, 2000), hAMC can be differentiated into hepatic-like cells. Differentiation of hAEC into neuronal-like (Sukuragawa, 2004) was reported. Functional genomic studies by Ming-Song Tsaia et al (2007) revealed that core gene expression profiles were preserved in those four kinds of MSC from amniotic fluid, amniotic membrane, cord blood and bone marrow. The core signature transcriptomes of all MSCs, in comparison with those of fetal organs, included genes involved in the regulation of extracellular matrix and adhesion, transforming growth factor-β receptor signaling, and the Wnt signaling pathways.
Mesenchymal stem cells of placenta: hAMSC and hCMSC are likely to originate from extraembryonic mesoderm. hAMSC were characterized in several publications (Surakagawa et al, 2004; int'Anker et al, 2004; Portmann-Lanz et al, 2006; Wolbank et al, 2007), though descriptions of hCMSC are very limited (Zhang et al, 2006). Profile of surface marker expression on hAMSC and hCMSc is close to bone marrow-derived MSC as both are positive for CD90 and CD105, but do not express markers of hematopoietic of leukocyte markers (Portmann-Lanz et al, 2006; Alviano et al, 2007; Zhao et al, 2005). Differentiation of hAMSC and hCMSC was demonstrated towards mesodermal lineages, while hAMSC could be induced to differentiate into neural (ectoderm) and pancreatic cells (endoderm) (Parolini et al, 2007). However, as hAMSC and hCMSC described in the above cited publications were derived by plastic-adherence techniques, they represent mixed populations and clones of multiple different stem and progenitor cells. Since these cells are not clonally-derived, their pluri- or multipotency remains unproven. Qualitative evidence of engraftment of chorionic and amniotic cells in newborn rat and swine was detected only by RT-PCR, without morphological evidence of engraftment and/or differentiation in tissues (Bailo et al, 2004).
Thus, though amniotic epithelial and amniotic mesenchymal stem cells have been studied in several reports over the past decade, information on chorionic mesenchymal and/or other stem cells has been not presented yet. Clonal-derived stem cell lines have not been described. Cell lines so far isolated from mesenchyme of placenta, have been characterized by limited capability of proliferation (only 5-10 passages). Mesenchymal cells from chorion have not been characterized by their ability to differentiate into cells of all three germ layers, nor have they been reported for their ability to engraft tissues of animals, differentiate in tissues, or form tumors. Further, placental tissue has not been yet reported for the presence of primitive pluripotent cells, different from mesenchymal stomal plastic-adherent cells.
The subject matter of this invention are novel stem cell populations originating from chorion of human placenta, termed chorionic colony-forming unit cells (CCFUC) which are uniquely characterized by expression of novel acetyl-coenzyme A binding protein (ACBD6) described in placental chorionic stromal stem cells by us (Soupene et al, 2008) and references therein.
Human mesenchymal stem cells of different origin have been a subject of multiple patents. Most of patents describe cells, capable of differentiation in connective tissue type, obtained from bone marrow, adhere to plastic, positive for surface markers SH2, SH3, SH4 (Caplan, U.S. Pat. No. 5,486,359 “Human mesenchymal stem cells”). Methods of directed differentiation of such stem cells ex vivo are further described (Bruder et al., U.S. Pat. No. 5,942,225 “Lineage-directed induction of human mesenchymal stem cell differentiation”). Methods and systems are described for selective expansion of target cell populations, and methods of treating patients with cell populations and products (Krauss et al., U.S. Pat. No. 6,338,942 “Selective expansion of target cell populations”), as well as methods of using non-autologous mesenchymal stem cells comprising treating a recipient (Bruder et al., U.S. Pat. No. 7,029,666 “Uses for non-autologous mesenchymal stem cells”). Simmons et al., describes mesenchymal precursor cells based upon expression of STRO-1 (U.S. Pat. No. 7,399,632 “Mesenchymal precursor cell”).
Placenta and its parts as a source of stem cells and physiologically active substances has been a subject of patents. Sanders (U.S. Pat. No. 3,862,002 “production of physiologically active placental substances”) teaches a system, in which viable placental tissue is placed in circulating culture medium and processed culture medium is then removed from tissue and desired substances are withdrawn. Limitations of this technique are based upon the fact that such method does not teach isolation of stem cell population. Also, such technique provides very low yield of cells as cells remain attached to tissue matrix.
Tseng (U.S. Pat. No. 6,326,019 “Grafts made from amniotic membrane”) teaches a method for making surgical grafts from amniotic membrane. As amnion represents only a very small fraction of placental tissue—less than 5%, yield of viable colony-forming unit cells from this specific source is very low. Low yield of primary cells requires multiple steps of cell passaging in order to obtain amount of cells required for therapeutic use in humans (about 100 million cells). Multiple passages in turn significantly increase the risk of chromosomal aberrations in cultured cells, making it highly risky for therapeutic use. Amniotic epithelial cells form teratomas and, therefore, can not be readily used in humans.
Cells isolated from umbilical cord—anatomical structure which connects a baby with placenta—have been described in several patents. Messina et al., (U.S. Pat. No. 7,524,489 “Regeneration and repair of neuronal tissue using postpartum-derived cells”), teaches method of treatment patients with cells derived from umbilical cord which do not express CD117 while expressing oxidized LDL receptort interleukin 8 or reticulon 1. Mistry et al., (U.S. Pat. No. 7,510,873 “Postpartum cells isolated from umbilical cord tissue, and methods of making and using the same”) teaches method of isolation of a cell from umbilical cord by enzymatic digestion that does not express CD117, CD31, CD34, CD141 or CD45 and express CD10, CD13, CD44, CD73, CD90, PDGFr-alpha or HLA-A, and further teaches use of these cells for treatment of retinitis (Mistry et al., U.S. Pat. No. 7,413,734 “Treatment of retinitis pigmentosa with human umbilical cord cells”). Harmon et al. (U.S. Pat. No. 7,560,276 “Soft tissue repair and regeneration, using postpartum-derived cells”) teaches use of these cells from umbilical cord and their products for soft tissue repair. Davies et al., U.S. Pat. No. 7,547,546 “Progenitor cells from Wharton's jelly of human umbilical cord” teaches obtaining cells from umbilical cord and their use in tissue repair. Use of proteolytic enzymes required for all the above described methods, first, dramatically reduces cell viability and yield of colony-forming unit cells, and, second, eliminates expression of many stem cell markers on cell surface, thus not allowing using sorting techniques for isolation of stem cells. The above described methods to obtain cells from umbilical cord feature same problems as obtaining stem cells from other low volume sources—yield of viable colony-forming unit cells from this source is very low. Low yield of primary cells requires multiple steps of cell passaging in order to obtain amount of cells required for therapeutic use in humans, while passaging increases the risk of chromosomal aberrations and tumor formation. Such risk of tumor formation from human fetal cells is more than real, and clinical cases of brain tumor development in patient from transplanted cells following administration of fetal cells were reported. It is, therefore, a subject matter of this invention to disclose novel techniques, methods and stem cell populations which could be obtained from the largest (95% of tissue mass of placenta) portion of placenta which allows to obtain very large yield of primary cells (several billion primary stem cells) without the use of proteolytic enzymes. Chen et al., (U.S. Pat. No. 7,534,606 “Placental stem cells and methods thereof”) claims a method of neurogenic differentiation of human placental stem cells, comprising culturing of placental stem cells in medium comprising an effective amount of 1-methyl-3-isobytylxantine.
Hariri (U.S. Pat. No. 7,045,148) reports that the first collection of blood from the perfused placenta, referred to as cord blood, contains populations of hematopoietic progenitor cells which are CD34 positive and CD38 positive or CD34 positive and CD38 negative or CD34 negative and CD38 positive. Subsequent perfusions of the placenta were reported to yield embryonic-like stem cells that are SSEA-3 negative, SSEA-4 negative, Oct-4 positive, ABC-p positive, CD10 positive, CD38 negative, CD29 positive, CD34 negative, CD44 positive, CD45 negative, CD54 positive, CD90 positive, SH2 positive, SH3 positive and SH4 positive. Hariri (U.S. Pat. No. 7,311,905 “Embryonic-like stem cells derived from post-partum mammalian placenta, and uses and methods of treatment using said cells”) describes a composition of human stem or progenitor cells that are positive for SH2, SH3, SH4 and Oct-4, while negative for CD34, CD45, SSEA3 and SSEA4, and obtained from placenta that has been drained from cord blood. Cells could express at least one of the following markers: CD10, CD29, CD44, CD54, CD90. Hariri (U.S. Pat. No. 7,468,276 “Placental stem cells”) describes the same placental stem cell population, adherent to plastic. Hariri (U.S. Pat. No. 7,255,879 “Post partum mammalian placenta, its use and placental stem cell populations”) teaches method to obtain the above described placental stem cell population by perfusing placenta via circulation with a perfusion solution containing an anticoagulant, growth factor or cytokine selected from a group consisting of a colony stimulating factor, interferon, erythropoietin, stem cell factor, thrombopoietin, an interleukin, granulocyte colony-stimulating factor, and any combination thereof, and collection of cells from perfusate. Methods of directed differentiation of these cells are described by Hariri (U.S. Pat. No. 7,498,171 “Modulation of stem and progenitor cell differentiation, assays and uses thereof”). The main disadvantage of methods described by Hariri in the above cited patents is in the fact that long-term perfusion of placenta is required to obtained claimed cells. It is know to those skilled in arts that in most cases of placentas obtained by Caesarian section and in all cases of placentas collected following natural birth, placentas are ruptured. This precludes the possibility of the long-term perfusion, as perfusate is rapidly lost via ruptures of chorion; therefore, long-term perfusion becomes unworkable. In most cases, arteries of umbilical cord rapidly completely constrict, and as thrombosis develops in placental vessels perfusion of placenta by techniques claimed by Haririr's patents (U.S. Pat. No. 7,045,148; U.S. Pat. No. 7,255,879; U.S. Pat. No. 7,311,905; U.S. Pat. No. 7,468,276) becomes practically impossible. Most importantly, perfusion of placenta via natural circulation does not allow collecting populations of stem cells which are located in stroma, interstitial tissue or non-perfused regions of placental circulation. Therefore, placental perfusion and cells claimed by Hariri's patents (U.S. Pat. No. 7,045,148; U.S. Pat. No. 7,311,905; U.S. Pat. No. 7,468,276; U.S. Pat. No. 7,498,171) allow obtaining very restricted and limited cell populations present in placenta, which belong to pool located inside the circulatory space. It is, therefore, a subject matter of this invention to disclose novel techniques, methods and stem cell populations which could be obtained without placental perfusion and collection of perfusate, as well as without the use of proteolytic enzymes. Placental stem cell populations and compositions of placental stem cells described by Hariri (U.S. Pat. No. 7,045,148; U.S. Pat. No. 7,311,905; U.S. Pat. No. 7,468,276) are characterized only by expression of several classical surface antigens (CD34, CD45, SSEA, SH-2, SH3, SH4, CD38, CD10, CD29, CD44, CD54, CD90). Moreover, Hariri wrongfully claims that cells described in these patents bear Oct-4 as surface marker (for example U.S. Pat. No. 7,311,905, claim 15), while Oct-4 is cytosolic/nuclear protein which is not expressed on surface of any cell, including embryonic stem cells. Placental cells claimed by Hariri (U.S. Pat. No. 7,045,148; U.S. Pat. No. 7,311,905; U.S. Pat. No. 7,468,276) are not characterized by their ability to form colonies, produce clones, differentiate into lineages of any of germ layers, form or not form teratomas, engraft immuno-compromised animals of humans, therefore differentiation potential and “sternness” of claimed cells is unknown. It is, therefore, a subject matter of this invention to disclose novel techniques, methods and stem cell populations from placenta which demonstrate specific phenotype and ability to form clones, differentiate into lineages of all 3 germ layers and engraft immunocompromized animals without formation of teratomas.
Naugtington et al. (U.S. Pat. No. 5,962,325) claims a method of making a composition comprising culturing fibroblast cells in 3-dimensional conditions. Naugtington et al. (U.S. Pat. No. 7,118,746 “Conditioned cell culture medium compositions and methods of use”) claims methods to obtain and use conditioned medium obtained from culture of human mesenchymal cells. The drawbacks of claimed methods is its limitations to secreted factors produced by mesenchymal cells, while stem cells produce different compositions of secreted factors which are more potent in tissue regeneration. Uchida (Uchida et al., 2000) described neurotrophic function of conditioned medium from human amniotic cells.